During the AO2023-1 expedition (1-21 June 2023), Melosira assemblages were collected using a handpump at a sampling site in Fram Strait. In addition to performing physiological measurements and filtering the material for trophic markers, living material was brought back to Oslo and two strains were established as unialgal cultures. The cultures are maintained in artificial seawater-based L1 medium at 4°C and 25 µmol m-2 s-1 light with a 16:8 L:D cycle. Lipids were extracted from freeze-dried samples following the method of Folch et al. (1957), using a dichloromethane/methanol mixture (2:1, v/v) in a sonication bath. The extracted lipids were converted to fatty acid methyl esters and analyzed by gas chromatography (Agilent 6890N Network GC, Agilent Technologies) equipped with a DB-FFAP capillary column (30 m length, 0.25 mm internal diameter, 0.25 μm film thickness), split injection, and a flame ionization detector. The temperature program ranged from 160°C to 240°C. Fatty acid analysis was conducted to complement datasets on fatty acid stable isotope compositions and sterol proportions. Collectively, the datasets enable a detailed characterization of the biochemical composition of different Melosira arctica-dominated algal assemblages from the Arctic Ocean, and a comparison with a unialgal culture of this important species.